# Patent

## Title
Methods, compositions and articles of manufacture for contributing to the treatment of cancers

## Abstract

Methods, compositions and articles of manufacture for contributing to the treatment of cancers, including solid tumors, are disclosed. The methods, compositions and articles of manufacture can utilize an endothelin B agonist (ETB) to enhance the delivery and resulting efficacy of a chemotherapeutic agent.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is a continuation and claims the benefit of priority pursuant to 35 U.S.C. §120 to U.S. patent application Ser. No. 12/390,376, filed Feb. 20, 2009, a continuation that claims priority pursuant to 35 U.S.C. §120 to U.S. patent application Ser. No. 11/461,961 filed Aug. 2, 2006, now abandoned, a continuation-in-part that claims priority pursuant to 35 U.S.C. §120 to U.S. patent application Ser. No. 11/360,236, filed Feb. 22, 2006, (which claims the benefit of priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Patent Application Nos. 60/655,656; 60/655,654; and 60/655,643, all filed on Feb. 22, 2005), a continuation-in-part that claims priority pursuant to 35 U.S.C. §120 to U.S. patent application Ser. No. 10/691,915, filed Oct. 23, 2003, now abandoned, a patent application that claims the benefit of priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 60/420,960, filed Oct. 24, 2002; each of which is hereby incorporated by reference in its entirety.


FIELD OF THE INVENTION
The present invention relates to methods, compositions and articles of manufacture for contributing to the treatment of cancers including solid tumors through administration of an endothelin agonist and a chemotherapeutic agent.
BACKGROUND OF THE INVENTION
Successful treatment of cancers, including solid tumors, remains an unfulfilled medical goal, despite increased understanding of the molecular biology of tumor cells and the availability of an increased number of potential therapeutic agents. For example, breast cancer incidence has increased substantially in the last 10 years, and is the single leading cause of death for women ages 40-49 years in the United States.
One problem in the treatment of cancers is that an effective dose of a wide variety of potential chemotherapeutic agents is restricted by these agents' non-selective, highly toxic effect on normal tissues. As a result, many patients suffer from the side effects of chemotherapy without reaping the benefits of the treatment. For example, the chemotherapeutic agent paclitaxel inhibits cellular proliferation and induces apoptosis of tumor cells. The clinical utility of paclitaxel has been hampered, however, by its dose limiting toxicities including hypersensitivity, neutropenia and peripheral neuropathy. Thus, there is a necessity to develop more specific and less toxic cancer therapies.
Targeted delivery of chemotherapeutic agents to tumors could have the advantage of enhancing the benefit of chemotherapeutic agents while minimizing their systemic toxic effects. Such targeted delivery could also serve to lower the required dose of chemotherapeutic agents thus potentially reducing the unacceptable adverse effects of these agents. One possible way to achieve targeted delivery of chemotherapeutic agents is to utilize the distinctive features of tumor vasculature.
Tumors greater than a few millimeters in size require a constant nutrient supply, and, therefore, develop their own vascular bed and blood flow. Folkman, Cancer Res, 46:467 (1986). Without constant nourishment from these developing blood vessels, the tumors become hypoxic and subsequently die. Recruitment of new vasculature from preexisting blood vessels is termed “angiogenesis.”
During angiogenesis, tumor blood vessels develop substantially differently from normal vasculature, and have different properties. Single layered epithelial cells are the first hastily formed tumor blood vessels. These newly formed tumor blood vessels do not have a smooth muscle layer or innervation. Tumors also incorporate mature blood vessels that possess all their autoregulatory functions. Mattsson et al., Tumor Blood Circulation, CRC Press, Boca Raton, pg. 129 (1979); Reinhold, Tumor Blood Circulation, CRC Press, Boca Raton, pg. 115 (1979); Warren, Tumor Blood Circulation, CRC Press, Boca Raton, pg. 26 (1979).
Vascular tone (the degree to which blood vessels are dilated or constricted) is governed by a host of endogenous factors including H+, K+, Ca2+, pO2, pCO2 and nitric oxide (NO), as well as other regulatory substances such as endothelin (ET-1). Secombe et al., Landes, Austin, pg. 40 (1994); Luscher et al., The endothelium: modulator of cardiovascular function, CRC Press, Boca Raton, pg. 61 (1990). ET-1 contributes significantly to regulating vascular tone (Yanagisawa et al., Nature, 332:411 (1988)) and investigators have shown an increase in ET1 and ETB receptor expression in solid tumors including breast carcinomas. Alanen et al., Histopathology, 36:161 (2000); Nelson et al., Cancer Res, 56:663 (1996); Kar et al., Biochem Biophys Res Commun 216:514 (1995); Pagotto et al., J Clin Invest, 96:2017 (1995); Yamashita et al., Cancer Res, 52:4046 (1992); Yamashita et al., Res Commun Chem Pathol Pharmacol, 74:363 (1991). Further, stimulation of ETB receptors causes an increase in blood supply to tumors through vasodilation of tumor blood vessels. The present invention takes advantage of this fact by using ETB receptor agonists to selectively increase blood flow to tumors to enhance the targeted delivery of chemotherapeutic agents.
SUMMARY OF THE INVENTION
The present invention is directed to the administration of endothelin agonists and a chemotherapeutic agent to contribute to the treatment of cancers including solid tumors. In particular, tumors have distinctive vasculature including an increased number of ETB receptors which, when bound, cause vasodilation. Because ETB receptors are vasodilators, an ETB receptor agonist, in combination with a chemotherapeutic agent, is useful in the treatment of a solid tumor, such as those found in breast cancers. The ETB receptor agonist can more effectively deliver chemotherapeutic agents to tumors resulting in enhanced treatment.
Specifically, one embodiment according to the present invention includes a method of contributing to the treatment of a cancer comprising administering an ETB agonist and a chemotherapeutic agent. In various embodiments of the methods according to the present invention, the ETB agonist and the chemotherapeutic agent can be administered substantially simultaneously or can be administered sequentially (with the chemotherapeutic agent administered prior to the ETB agonist or the ETB agonist administered prior to the chemotherapeutic agent). In certain embodiments according to the present invention when the ETB agonist and the chemotherapeutic agent are administered substantially simultaneously, they can be administered as a single composition.
Another embodiment according to the present invention includes a composition comprising a chemotherapeutic agent, an ETB agonist, and an optional excipient. Another embodiment according to the present invention includes an article of manufacture comprising a composition comprising an ETB agonist, and instructional information directing the administration of the composition with a chemotherapeutic agent to treat a solid tumor. Articles of manufacture according to the present invention can further comprise one or more chemotherapeutic agents. When articles of manufacture according to the present invention include one or more chemotherapeutic agents, the ETB agonist and the chemotherapeutic agent can be part of the same composition, can be provided as separate compositions, or both.
Cancers that are treated with the methods, compositions or articles of manufacture according to the present invention can include solid tumors including, without limitation, ovarian tumors, colon tumors, Kaposi's sarcoma, breast tumors, melanoma, prostate tumors, meningiomas, liver tumors, breast phyllode tumors and combinations thereof.
Endothelin B agonists used in accordance with the methods, compositions or articles of manufacture of the present invention can selectively increase blood supply to solid tumors thus increasing the delivery of chemotherapeutic agents to the solid tumor. Endothelin B agonists that can be used in accordance with the present invention can include, without limitation, one or more of ET-1, ET-2, ET-3, BQ3020, IRL1620 (N-suc-[Glu9, Ala11,15]ET-1 (8-21)), sarafotoxin 56c, [Ala1, 3, 11, 15]ET-1, and combinations thereof. Chemotherapeutic agents can include, without limitation, one or more of adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, 5-fluorouracil, and combinations thereof. Particular methods, compositions or articles of manufacture according to the present invention will include IRL1620 as the ETB agonist with a chemotherapeutic agent selected from the group consisting of paclitaxel, doxorubicin, 5-fluorouracil, and combinations thereof.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the effect of IRL1620 on paclitaxel-induced changes in tumor perfusion;
FIGS. 2A-2E show the effect of ET-1 on systemic hemodynamics of cancer-free and breast tumor-bearing rats;
FIGS. 3A-3B show the effect of ET-1 on blood flow and regional vascular resistance in the breast tissue of cancer-free and breast tumor-bearing rats;
FIGS. 4A-4C show the effect of ET-1 on perfusion, concentration of moving blood cells (CMBC), and velocity of blood cells in breast tissue of cancer-free and breast tumor-bearing rats;
FIGS. 5A-5C show the effect of BQ788 on ET-1-induced changes in blood perfusion, CMBC, and velocity of blood cells in breast tissue of cancer-free and breast tumor-bearing rats;
FIG. 6 shows the effect of vehicle or IRL1620 on plasma pharmacokinetics of paclitaxel analysis in normal and tumor bearing rats as determined by HPLC;
FIGS. 7 and 8 show the effect of vehicle or IRL1620 on plasma pharmacokinetics of [3H]-paclitaxel as determined by liquid scintillation counting;
FIGS. 9A and 9B show the effect of IRL1620 on breast tumor perfusion as measured by Laser Doppler Flowmetry;
FIG. 10 shows the time dependent effect of IRL1620 administration on [3H] paclitaxel concentration in tumor and major organs of breast tumor bearing rats;
FIG. 11 shows the percentage difference in the body weight of breast tumor bearing rats compared to the beginning of treatment;
FIG. 12 shows the effect of IRL1620 administration on the tumor volume of breast tumor bearing rats;
FIG. 13 shows the effect of IRL1620 administration on tumor progression, stasis and regression in breast tumor bearing rats;
FIGS. 14A and 14B show the effects of different doses or IRL1620 on prostate tumor perfusion as measured by Laser Doppler Flowmetry (14A) and the percent change in perfusion of prostate tumor from baseline following administration of IRL1620 (14B);
FIG. 15 shows the effect of IRL1620 on [14C]-doxorubicin (DOX) concentration in tumor and other major organs of prostate tumor bearing rats;
FIG. 16 shows the body weight (16A); tumor volume (16B); and tumor weight (16C) of prostate tumor bearing rats following administration of IRL1620 and DOX;
FIG. 17 shows the body weight (17A); tumor volume (17B); and tumor weight (17C) of prostate tumor bearing rats following administration of IRL1620 and 5-Fluorouracil (5-FU);
FIGS. 18A and 18B show the effect of IRL1620 on melanoma tumor perfusion as measured by Laser Doppler Flowmetry (18A) and the percent change in perfusion of melanoma tumor from baseline following administration of IRL1620 (18B);
FIG. 19 shows the effect of IRL1620 on [3H]-paclitaxel concentration in tumor and other major organs of melanoma tumor bearing rats.



DETAILED DESCRIPTION
I. Definitions
Instructional Information: As used herein, the term “instructional information” shall mean material accompanying a pharmaceutical product that provides a description of how to administer the product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding use of the product. This instructional information generally is regarded as the “label” for a pharmaceutical product. Instructional information can come in many forms including, without limitation, a paper insert, c.d. rom or directions to a web site containing information relating to the pharmaceutical product.
Prodrug: As used herein, the term “prodrug” shall mean compounds that transform rapidly in vivo to a compound useful in the invention, for example, by hydrolysis. A thorough discussion of prodrugs is provided in Higuchi et al., Prodrugs as Novel Delivery Systems, Vol. 14, of the A.C.S.D. Symposium Series, and in Roche (ed.), Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987.
Treat, Treatment or Contributing to the Treatment Of: As used herein, the terms “treat”, “treatment” and “contributing to the treatment of” shall mean preventing, retarding the progression or growth of, shrinking, or eliminating a cancer including a solid tumor. As such, these terms include both medical therapeutic and/or prophylactic administration, as appropriate.
Substantially Simultaneously: As used herein, the term “substantially simultaneously” shall mean that two pharmaceutical preparations (i.e. an ETB agonist and a chemotherapeutic agent) are administered at the same time. According to this definition, “same time” should be read to include exactly simultaneously as well as within about ten minutes.
Most chemotherapeutic agents have cytotoxic properties that are targeted to destroy cancer cells, but in the process inflict considerable damage to the body's normal physiological systems. It would be of great advantage, therefore, to selectively deliver chemotherapeutic agents to solid tumors thus helping to avoid these negative effects of cancer treatment.
The angioarchitecture of tumor blood vessels is different from that of normal blood vessels. Carmeliet & Jain, Nature, 407:249 (2000). Therefore, the vascular reactivity of tumors differs from that of normal tissue. For example, the administration of nitric oxide donors, nicotinamide and bradykinin agonists modulate blood flow to tumors. Jordan et al., Int J Radiat Oncol Biol Phys, 48:565 (2000); Fukumura et al., Am J Pathol, 150:713 (1997); Hirst et al., Br J Radiol, 67: 795 (1994).
Endothelin is a vasoactive substance that modulates blood flow and is present in large concentrations in breast carcinoma tissues compared to normal breast tissue (specifically, endothelin can be present in an amount of about 12 pg/mg in breast carcinoma tissues as compared to about 0.12 pg/mg in normal breast tissue). Kojima et al., Surg Oncol, 4(6):309 (1995); Kurbel et al., Med Hypotheses, 52(4):329 (1999); Patel et al., Mol Cell Endocrinol, 126(2):143 (1997); Yamashita et al., Cancer Res, 52(14):4046 (1992); Yamashita et al., Res Commun Chem Pathol Pharmacol, 74(3):363 (1991). Endothelins are a family of cyclic peptides with 21 amino acids, comprising three isoforms in mammals, ET-1, ET-2 and ET-3. Inoue et al., Proc Natl Acad Sci USA 86:2863 (1989); Yanagisawa et al., Nature, 332:411 (1988). Endothelins exert their effects by binding to two distinct cell surface receptors, ETA and ETB. The ETB receptor binds the three peptide isotypes with equal affinity. In contrast, the ETA receptor binds ET-1 with higher affinity than the other isoforms. Both receptors belong to the G protein-coupled receptor system and mediate biological responses from a variety of stimuli, including growth factors, vasoactive polypeptides, neurotransmitters and hormones. Masaki, J Cardiovasc Pharmacol, 35:S3 (2000); Gulati, Preface. Adv Drug Deliv Rev, 40:129 (2000); Gulati et al., Am J Physiol, 273:H827 (1997); Levin, N Engl J Med, 333:356 (1995). ETB receptors, a focus of the present invention, are present on both endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) and are increased in breast cancer tissue (including in invasive as well as in ductal and lobular breast carcinoma tissue in humans) when compared to normal breast tissue. Wulfing et al., Oncol Rep, 11:791 (2004); Wulfing et al., Clin Cancer Res, 9:4125 (2003); Alanen et al., Histopathology, 36(2):161 (2000). Endothelin acts on ETB receptors to produce vascular dilation and increase blood flow to breast tumor tissue. ETB receptors predominating on ECs, produce vasodilatation via the release of factors such as prostacyclin and nitric oxide. de Nucci et al., Proc Natl Acad Sci USA, 85:9797 (1988). Because ET-1 produces an increase in blood flow to tumors by stimulating ETB receptors, an ETB receptor agonist can be used to selectively increase blood supply to tumors, thus increasing the targeted delivery and resulting efficacy of chemotherapeutic agents.
ETB receptors have been shown in, for example and without limitation, ovarian cancers, myofibroblasts, Kaposi's sarcoma tumor and intratumoral vessels, breast cancers and melanomas. Bagnato et al., Am J Pathol, 158:841 (2001); Alanen et al., Histopathology, 36(2):161 (2000); Bagnato et al., Cancer Res, 59:720 (1999); Kikuchi et al., Biochem Biophys Res Comm, 219:734 (1996). Therefore, administration of an ETB receptor agonist in combination with a chemotherapeutic agent can be used to contribute to the treatment of solid tumors, including, without limitation, ovarian cancer, colon carcinoma, Kapoli's sarcoma, breast cancer, and melanomas.
ETB agonists useful in accordance with the present invention include, without limitation, ET-1, ET-2, ET-3, BQ3020, IRL1620 (N-suc-[Glu9, Ala11,15]ET-1 (8-21)), sarafotoxin 56c, [Ala1, 3, 11, 15]ET-1, and combinations thereof. [Ala1,3,11,15]ET-1 is a linear analog of ET-1 in which the disulfide bridges have been removed by substitution of Ala for Cys residues. Saeki et al., Biochem Biophys Res Commun, 179:286 (1991). BQ3020 and IRL1620 are truncated linear synthetic analogs of ET-1 and are the most widely used selective synthetic agonists. IRL1620 is a linear ET-analog whose structure is based on the carboxy terminal end of ET-1 and has 120,000 fold selectivity for the ETB receptors. Okada & Nishikibe, Cardiovasc Drug Rev, 20:53 (2002); Douglas et al., Br J Pharmacol, 114:1529 (1995). IRL1620 is a highly selective and potent ETB agonist, with evidence being reported of its selectivity for the ETB1 receptor subtype in preference over the ETB2 subtype. Brooks et al., J Cardiovasc Pharmacol, 26 Suppl 3:S322 (1995).
Chemotherapeutic agents useful in accordance with the present invention include, for example and without limitation, alkylating agents, antimetabolites, hormones and antagonists thereof, radioisotopes, antibodies, as well as natural products, and combinations thereof. For example, an ETB agonist can be administered with antibiotics, such as doxorubicin and other anthracycline analogs, nitrogen mustards, such as, without limitation, cyclophosphamide, pyrimidine analogs such as, without limitation, 5-fluorouracil, cisplatin, hydroxyurea, and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the ETB agonist can be administered in conjunction with, without limitation, leuprolide or goserelin (synthetic peptide analogs of LH-RH). Additional non-limiting examples of chemotherapeutic agents that can be used with the present invention include adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, interferon (alpha, beta, and/or gamma), interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, and therapeutically effective analogs and derivatives of the same.
In one embodiment of the present invention, an endothelin agonist is used in conjunction with a chemotherapeutic agent to contribute to the treatment of a solid tumor. In this method, the endothelin agonist, notably an ETB agonist, increases blood flow to the tumor, which is rich in ETB receptors. The ETB agonist, therefore, provides a more selective target for the chemotherapeutic agent and improves the chemotherapeutic effect of the agent.
It is theorized, but not relied upon herein, that endothelin agonists stimulate ETB receptors to dilate tumor blood vessels, thereby increasing blood flow and the resultant delivery of chemotherapeutic agents to the tumor. The increased blood perfusion of tumors caused by endothelin agonists also increases oxygenation of the tissue. Improved oxygenation can enhance the therapeutic action of chemotherapeutic agents. Endothelin also can have mitogenic properties. The mitogenic actions of endothelin can help increase the action of chemotherapeutic agents, when administered together. The mitogenic action of an endothelin agonist can increase the action of chemotherapeutic agents by improving their incorporation into dividing cells, thus increasing their efficacy.
Chemotherapy is frequently indicated as an adjuvant to surgery in the treatment of a cancer. The goal of chemotherapy in the adjuvant setting is to reduce the risk of recurrence and enhance disease-free survival when the primary tumor has been controlled. Chemotherapy is utilized as a treatment adjuvant for a cancer, frequently when the disease is metastatic. An ETB agonist, therefore, is particularly useful before or following surgery in the treatment of a solid tumor in combination with chemotherapy.
BREAST TUMOR MODEL
Example 1
Effect of IRL1620 and Paclitaxel on Breast Tumor Perfusion
The following studies were conducted to examine the systemic hemodynamics and regional circuitry effects of ET-1 in normal and breast tumor-bearing rats.
One extensively studied breast tumor model is the chemically induced rat mammary carcinogenesis model. van Zwieten, The rat as animal model in breast cancer research. Martinus Nijhoff Publishers, Boston, pg. 206 (1984); Dao et al., J Natl Cancer Inst, 71:201 (1983); Russo et al., J Natl Cancer Inst, 61:1439 (1978); Huggins et al., Science, 137 (1962); Huggins et al., Proc Natl Acad Sci USA, 45:1294 (1959). Chemically induced mammary tumorigenesis in rats is the model most closely resembling a human cancer. Russo et al., Lab Invest, 62:244 (1990). In terms of tissue architecture, the mammary gland of a rat is comparable to that of human women. It is formed by an epithelium that covers the ducts and alveoli and a stroma, the connective tissue scaffolding of this organ. These two compartments are in continuous interaction during embryonic development and throughout adulthood. Therefore, this autochthonous experimental model was selected as a model in the presently described studies as it most closely resembles human cancer. Id.
Chemically induced rat mammary carcinogenesis typically is achieved by administration of 7,12-dimethylbenzene(a)anthracene (DMBA) or N-methylnitrosourea (MNU). Rogers et al., Chemically induced mammary gland tumors in rats: modulation by dietary fat. Alan R. Liss, Inc., New York 255 (1996). Tumors induced by DMBA or MNU have different morphological characteristics. In particular, tumors induced by MNU are more localized at the breast and are less likely to metastasize. Macejova et al., Endocr Regul, 35:53 (2001). Therefore, MNU often is chosen as the chemical agent for the specific induction of breast tumors in rats. These breast tumors can be benign with fibroadenomas and papillomas, or they can be malignant. van Zwieten, Martinus Nijhoff Publishers, Boston, pg. 206 (1984). Rats have six pairs of mammary glands, one in the cervical region, two in the thoracic region, one in the abdominal region, and two in the ingual region. Id.; Astwood et al., Am J Anat, 61 (1937). Virgin rats treated with MNU develop more tumors in the thoracic region than the abdominal region. Russo et al., Lab Invest, 57:112 (1987).
Female Sprague Dawley rats (Harlan Co., Madison, Wis.) weighing 180-200 grams (g) were used. All animals were housed, three to a cage, in a temperature controlled room (23±1° C.), humidity (50±10%), and artificial light (0600-1800 hr). The animals were given food and water ad libitum. The experiments were conducted after the animals had been acclimatized to the environment for at least four days.
N-methylnitrosourea (MNU) was purchased from Ash Stevens Inc. (Detroit, Mich.). IRL1620 and Endothelin-1 (ET-1) were obtained from American Peptide Company Inc. (Sunnyvale, Calif.). ET-1 was dissolved in 0.1% albumin.
MNU (50 mg/kg) or saline (1 ml/kg) was administered intraperitoneally (i.p.) to the female Sprague Dawley rats. After tumors reached about 2-4 cm in diameter, blood flow experiments were performed.
During blood flow experiments, rats were anesthetized with urethane (1.5 g/kg, i.p.) (Sigma Chemicals, St. Louis, Mo.), and the left femoral vein was cannulated (PE 50 tubing, Clay Adams, Parsipanny, N.J.) for drug administration.
Animals were divided into the following groups:

    
    
        Group I: Saline+paclitaxel (taxol; 3 mg/kg; 15 minutes after saline administration) in normal rats (N=4);
        Group II: IRL1620 (3 nmol/kg)+paclitaxel (3 mg/kg; 15 minutes after IRL1620 administration) in normal rats (N=4);
        Group III: Saline+paclitaxel (3 mg/kg; 15 minutes after saline administration) in tumor bearing rats (N=4); and
        Group IV: IRL1620 (3 nmol/kg)+paclitaxel (3 mg/kg; 15 minutes after IRL1620 administration) in tumor bearing rats (N=4).
    
    


Blood perfusion to the mammary gland of the rats was measured using laser Doppler flowmetry. See Song et al., Int J Radiat Oncol Biol Phys, 18:903 (1990); Song et al., Int J Radiat Oncol Biol Phys, 17:1041 (1989). In this procedure, the animals were shaved around the nipples and the skin surrounding the mammary glands was dissected out. A standard model fiber optic probe was secured to the mammary artery and connected to a Periflux PF2b 4000 Laser Doppler Flowmetry (Perimed KB, Stockholm, Sweden). The time constant was set to 1.5 seconds, and the band width was set to 4 KHz. Data were analyzed using analysis of variance (ANOVA) followed by Duncan's test. A level of p<0.05 was considered significant.
No change in blood flow to the breast tissue of normal rats was observed following the administration of saline or IRL1620 and paclitaxel. Significant differences were observed between the blood flow in tumor tissue after IRL1620 injection (36.3%, p<0.05) and after paclitaxel following IRL1620 administration (51.9%, p<0.0.5) from baseline (see FIG. 1). This study thus demonstrates that IRL1620 can provide an important adjuvant to cancer treatments including the administration of chemotherapeutic agents.
Example 2
Effect of ET-1 Infusion on Systemic Hemodynamics and Blood Flow to the Mammary Tissue of Normal and Tumor-Bearing Rats
MNU and saline treatments were performed as i.p. injections three months prior to the studies. Rats were palpated regularly starting four weeks after the treatments. Once tumors reached about 4-8 mm in diameter, experiments were initiated.
Rats were anesthetized with urethane (1.5 g/kg, i.p.) (Sigma Chemicals, St. Louis, Mo.). All surgical areas were shaved and cleaned with alcohol swabs. The left femoral vein was cannulated (PE 50 tubing, Clay Adams, Parsipanny, N.J.) for drug administration. The left femoral artery was cannulated (PE 50 tubing) and was used for withdrawal of reference blood sample in microsphere studies using a withdrawal pump (Model 22, Harvard Apparatus, South Natick, Mass.). The right femoral artery was cannulated (PE 50 tubing) and connected to a Gould P23 ID pressure transducer for recording the blood pressure on a Grass P7D polygraph (Grass Instrument Co., Quincy, Mass., USA) through a 7PI preamplifier. The heart rate (HR) was recorded through a 7P4B Grass tachograph (Grass Instrument Co., Quincy, Mass.) triggered from blood pressure signals. The right carotid artery was exposed and a PE 50 tubing was guided through the common carotid artery into the left ventricle. The presence of the cannula in the left ventricle was confirmed by recording the pressure on the Grass polygraph using the Statham P23 DC pressure transducer (Grass Instrument Co., Quincy, Mass.). When the cannula reached the left ventricle; the diastolic pressure dropped to zero. In order to maintain the blood pO2, pCO2, and pH constant, and to avoid the effect of respiration on blood pressure and HR, animals were kept on constant rate artificial respiration by inserting an endotracheal cannula connected to a rodent ventilator (Model 683, Harvard Apparatus Inc., South Natick, Mass.).
Rats were initially divided into two groups, each receiving one of the following treatments:

    
    
        Group I: ET-1 (50 ng/kg/min) infusion for 30 minutes in rats treated with saline (normal rats) (N=6); and
        Group II: ET-1 (50 ng/kg/min) infusion for 30 minutes in treated with MNU (50 mg/kg, i.p.; tumor rats) (N=6).
    
    


Systemic hemodynamic and regional circulation parameters were determined at baseline, 30, 60, and 120 minutes after starting ET-1 (50 ng/kg/min) infusion. Because ET-1 infusion was performed for 30 minutes, the 30-minute data shows the effect of ET-1, and the 60- and 120-minute data indicates duration of the ET-1 effect.
Systemic hemodynamics and regional blood circulation were determined using a literature described procedure. See Gulati et al., J Lab Clin Med, 126:559 (1995); Gulati et al., Life Sci., 55:827 (1994); Sharma et al., Artif Cells Blood Substit Immobil Biotechnol, 22:593 (1994). At each measurement, a thoroughly mixed suspension of approximately 100,000 microspheres (15±1 μm diameter) labeled with 46Sc (scandium), 113Sn (tin), 141Ce (cerium), or 95Nb (niobium) (New England Nuclear Corporation, Boston, Mass., USA) in 0.2 ml saline were injected into the left ventricle and flushed with 0.3 ml saline over a 15 second period. In order to calculate blood flow, arterial blood was withdrawn at a rate of 0.5 ml/min through the right femoral artery. Blood was withdrawn for 90 seconds starting about 5-10 seconds before microsphere injection.
Blood perfusion to the mammary gland of the rats was measured using laser Doppler flowmetry. See Song et al., Int J Radiat Oncol Biol Phys, 18:903 (1990); Song et al., Int J Radiat Oncol Biol Phys, 17:1041 (1989). The animals were shaved around the nipples. The skin surrounding the mammary glands was dissected out as a lambeau about 6 cm wide and about 4 cm long. A standard model fiber optic probe was applied to the surface of the lambeau, and secured to the tissue by double stick tape. The lambeau was placed in a metal holder and taped down to prevent movement, then connected to a Periflux PF2b 4000 Laser Doppler Flowmetry (Perimed KB, Stockholm, Sweden). The time constant was set at 1.5 seconds and the bandwidth was set at 4 KHz. Data were analyzed using analysis of variance followed by Duncan's test. A level of p<0.05 was considered significant.
At the end of the experiment, animals were sacrificed with an overdose of pentobarbital sodium. All tissues and organs were dissected out, weighed, and placed in vials. The radioactivity in the standards, the blood samples, and the tissue samples were counted in a Packard Minaxi Auto-Gamma 5000 series gamma counter (Packard Instruments Co., Downers Grove, Ill.) with preset windows discriminating the isotope energies. The following parameters were calculated: (1) cardiac output (CO) ((radioactivity injected×withdrawal rate of arterial blood)/radioactivity in sampled arterial blood), (2) stroke volume (SV) (CO/HR), (3) total peripheral resistance (TPR) (mean arterial pressure (MAP)/CO), (4) regional blood flow ((radioactivity in tissue×withdrawal rate of arterial blood)/radioactivity in sampled arterial blood), and (5) regional vascular resistance (MAP/regional blood flow). The data were calculated using computer programs described in the literature. Saxena et al., Comput Programs Biomed, 12:63 (1980).
The baseline systemic hemodynamic parameters in normal (saline treated) rats were MAP: 111.1±4.8 mmHg; CO:268.6±17.6 ml/min; SV:0.87±0.06 ml; TPR:419.6±24.37 mmHg·min/ml; and HR:312.5±20.2 beats/min. In normal rats, a significant increase in MAP was observed at 30 minutes (14.5%; p<0.05), and a decrease at 120 minutes (17.8%; p<0.05) following ET-1 infusion. TPR increased at 120 minutes (49.2%; p<0.05). CO decreased at 60 and 120 minutes (22.9% and 42.5% respectively; p<0.05) after ET-1 infusion. SV decreased at 60 and 120 minutes (20.9% and 36% respectively; p<0.05). No significant change in HR was observed (FIGS. 2A-2E).
The baseline systemic hemodynamic parameters in tumor-bearing (MNU treated) rats were similar to that in normal rats. A significant increase in MAP was observed at 30 minutes (19.1%; p<0.05) and at 60 minutes (15.3%; p<0.05) following ET-1 infusion in tumor-bearing rats. TPR increased at 30 minutes (73.9%; p<0.05), 60 minutes (39.7%; p<0.05), and 120 minutes (71.4%; p<0.05) following administration of ET-1. CO decreased at 30, 60 and 120 minutes (29.4%, 16.7% and 36.1% respectively; p<0.05). SV decreased significantly at 30, 60 and 120 minutes (31.1%, 17.9% and 32.1% respectively; p<0.05). No change in HR was observed (FIGS. 2A-2E).
No significant change in blood flow to the breast tissue or change in vascular resistance of normal saline-treated rats was observed following the administration of ET-1. Significant differences were observed between the blood-flow and the regional vascular resistance in the breast tissue of tumor-bearing (MNU treated) when compared to normal (saline treated) rats. A significant increase (153%; p<0.05) in blood flow to the breast tissue of tumor-bearing rats as compared to normal rats was observed at 60 minutes following administration of ET-1. The vascular resistance in the tumor-bearing rats was significantly different at baseline (102%; p<0.05) and at 60 minutes (147%; p<0.05) following ET-1 administration compared to normal rats (FIGS. 3A-3B).
FIGS. 4A-4C show the changes in perfusion, concentration of moving blood cells (CMBC), and velocity of red blood cells (RBC) in the breast tissue of tumor-bearing and normal rats. Blood perfusion in the breast tissue of normal rats did not significantly change after ET-1 administration. Perfusion in the breast tissue of tumor-bearing rats at 30 minutes following ET-1 administration increased significantly (176%; p<0.05) compared to normal rats. This increase in perfusion returned to baseline at 60 and 120 minutes following ET-1 administration in tumor-bearing rats.
The CMBC in tumor-bearing rats increased significantly (54%; p<0.05) at 60 minutes post ET-1 administration as compared to normal rats. CMBC returned to baseline at 120 minutes after ET-1 administration. The velocity of RBC increased significantly (252%; p<0.05) at 30 minutes post ET-1 administration compared to normal rats. Two hours (120 minutes) after ET-1 administration, the velocity of RBC in tumor-bearing rats returned to baseline (FIGS. 4A-4C).
Another study evaluated the role of ETB receptors on the changes induced by ET-1 infusion on the systemic hemodynamics and blood flow to the mammary tissue of normal rats and rats with breast tumors. BQ788 (i.e., N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyll-eucyl-D-1-methoxycarbonyltrptophanyl-D-Nle) is a specific ETB receptor antagonist that inhibits binding to ETB receptors with an IC50 value of 1.2 nM. BQ788 was therefore used to determine the role of ETB receptors in ET-1 induced vasodilation in the breast tumor. This study employed the methods described in the previous study except that animals were divided into the following groups:

    
    
        Group I: BQ788 (American Peptide Company Inc. (Sunnyvale, Calif.) dissolved in saline at 0.5 pmol/kg) infusion for 20 minutes followed by ET-1 (50 ng/kg/min) infusion for 30 minutes in normal saline-treated rats (N=5); and
        Group II: BQ788 (0.5 pmol/kg) infusion for 20 minutes followed by ET-1 (50 ng/kg/min) infusion for 30 minutes in tumor-bearing MNU-treated rats (50 mg/kg, i.p.) (N=5).
    
    


FIGS. 5A-5C show the effect of BQ788 on changes induced by ET-1 in blood perfusion, CMBC, and velocity of RBC in tumor-bearing and normal rats, respectively. Blood perfusion in the breast tissue of normal rats did not change significantly after BQ788 administration or ET-1 infusion. However, perfusion in the breast tumor tissue of tumor-bearing rats decreased significantly at 30 (25.25.+−0.5.7%; P<0.05) and 60 minutes (25.17.+−0.2.8%; P<0.05) following ET-1 infusion in BQ788 pretreated rats. Pretreatment with BQ788 attenuated the increase in perfusion induced by ET-1 in tumor-bearing rats. No difference between the perfusion in breast tissue of tumor-bearing rats and normal rats was observed following ET-1 administration in BQ788 pretreated rats. This result suggests that ET-1-induced vasodilatory responses are mediated through ETB receptors.
The baseline CMBC in tumor-bearing rats was significantly higher than the baseline CMBC of breast tissue of normal rats (42.4%; P<0.05). However, after BQ788 infusion, no difference between CMBC of tumor-bearing and normal rats was observed. In addition, no difference in velocity of RBC between the two groups was observed (FIGS. 5A-5C).
The above tests show the effect of ET-1 on systemic hemodynamics and blood flow to the breast tissue of saline-treated and MNU-treated tumor-bearing rats. It is known that ET-1 stimulates angiogenesis by promoting production of VEGF. Studies have shown that ET-1 is increased in many cancer tissues like breast carcinoma (Yamashita et al., Res Commun Chem Pathol Pharmacol, 74:363 (1991)), breast phyllode tumor (Yamashita et al., Cancer Res, 52:4046 (1992)), prostate carcinoma (Nelson et al., Cancer Res, 56:663 (1996)), liver carcinoma (Kar et al., Biochem Biophys Res Commun 216:514 (1995)), and some meningiomas (Pagotto et al., J Clin Invest, 96:2017 (1995)). The above tests demonstrate changes in ET-1-induced vascular responses in the breast tumor. The method used in these tests was a well-established radioactive microsphere technique to study the systemic hemodynamics and regional blood circulation. Gulati et al., Am J Physiol, 273:H827 (1997); Gulati et al., Crit Care Med, 24:137 (1996); Gulati et al., J Lab Clin Med, 126:559 (1995); Gulati et al., Life Sci, 55:827 (1994).
Example 3
Effect of IRL1620 on Pharmacokinetics of Paclitaxel
Altering blood flow dynamics in the body can significantly affect the pharmacokinetics of a therapeutic moiety, and paclitaxel is known to have complex pharmacokinetic properties. See, for example, Sparreboom et al., Cancer Res 56:2112 (1996a); Gianni et al., J Natl Cancer Inst 87:1169 (1995b); Sonnichsen & Relling, Clin Pharmacokinet 27:256 (1994); Huizing et al., J Clin Oncol 11:2127 (1993); Brown et al., J Clin Oncol 9: 1261 (1991); Longnecker et al., Cancer Treat Rep 71:53 (1987); Wiemik et al., Cancer Res 47:2486 (1987b). It is therefore important to understand the impact of IRL1620 on the plasma pharmacokinetics of paclitaxel. The presently described study was therefore conducted to determine whether IRL1620, a selective ETB receptor agonist, alters the pharmacokinetics of paclitaxel in breast tumor bearing rats.
Virgin female Sprague Dawley rats (Harlan Co., Madison, Wis.), 48 days old (120-140 g) were used for this study. Upon arrival, all rats were housed three to a cage, in a room with controlled temperature (23±1° C.), humidity (50±10%) and artificial light (0600-1800 hr). The rats were given food and water ad libitum. The experiments were begun only after the rats have been acclimatized to the environment for at least 4 days.
IRL1620 was purchased from Sigma-Aldrich (St. Louis, Mo.). Paclitaxel (6 mg/mL solution) was purchased from Ben Venue Laboratories Inc. (Bedford Ohio). Ketamine and xylazine were purchased from Phoenix Scientific, Inc. (St. Joseph, Mo.). [3H]-paclitaxel (ImCi, 6.4 Ci/mmol, specific activity) was purchased from Moravek Biochemicals (Moravek Biochemicals, CA). Urethane was purchased from Sigma Aldrich (Sigma Chemicals, St. Louis, Mo.).
N-methyl-n-nitrosourea (MNU) was administered at a dose of 50 mg/kg, i.p. and rats were palpated twice weekly. Once tumors reached about 75-100 mm3, pharmacokinetic studies were performed.
HPLC-UV Studies.
Rats were anesthetized with a single i.p. injection of urethane (1.5 mg/kg) (Sigma Chemicals, St. Louis, Mo.). The right femoral region was shaved and cleaned with surgical disinfectant and alcohol. The right femoral artery and vein were exposed and cannulated with sterile PE-50 tubing. The neck was shaved and cleaned with surgical disinfectant and alcohol. A middle incision was made around the neck region and the trachea was intubated and connected to a rodent ventilator (Model 683, Harvard Apparatus Inc., South Natick, Mass.). All surgeries were performed under aseptic conditions. Neosporin antibiotic cream (Pfizer, Morris Plains, N.J.) was applied to the wounds to prevent infection. A 45-minute recovery period was given before drug administration.
Normal (saline-treated) and tumor bearing (MNU-treated) rats were used. Paclitaxel was given i.v. (3 mg/kg) 15 minutes after IRL1620 (3 nmol/kg) or vehicle (saline, 3 mL/kg) administration. Blood was collected before IRL1620 administration to provide baseline values. 0.5 mL of blood was drawn from the rats in heparinized syringes at baseline, 5, 30 minutes, and 2, 6, and 10 hours after paclitaxel administration. The samples were centrifuged and plasma was harvested and stored at −80° C. until analysis.
Plasma samples were analyzed for paclitaxel using an HPLC system. Briefly, plasma was thawed and mixed with 50 μL of the internal standard N-cyclohexyl benzamide (3 mM, lower standard curve and 30 mM, higher standard curve) and 3 mL of ethyl ether (Fisher Scientific, Chicago, Ill.) in a 13×100 glass culture tube. The mixture was shaken using a reciprocal shaker for 5 minutes and then centrifuged for 5 minutes at 3,000 rpm at 4° C. The resulting supernatant was transferred to a 13×100 borosilicate glass culture tube and evaporated under a stream of nitrogen in a heated water bath (37° C.). The residue was reconstituted with 200 μL of mobile phase A (50% deionized water, 50% acetonitrile). A 100 μL (lower standard curve and samples collected after IV administration) aliquot of the reconstituted material was injected into a 4 mm NovaPak 150×3.9 mm C18 column (Waters Associates, Milford, Mass.) preceded by a 4 mm NovaPak 20×3.9 mm C18 precolumn using a Waters 2695 separations module connected to a Waters 2487 absorbance detector set at 227 nm. A linear gradient was started with 100% mobile phase A pumped at a flow rate of 1 mL/min. Mobile phase A was then decreased to 70% from 10 to 11 minutes with mobile phase A maintained at 70% from 11 to 16 minutes to remove materials slowly eluting from the column before the next injection. Subsequently, mobile phase A was increased to 100% from 16 to 17 minutes and maintained at 100% mobile phase A for three minutes providing a total run time of 20 minutes. Plasma concentrations for paclitaxel were calculated from the ratio of the area of the paclitaxel peak to the area of the N-cyclohexyl benzamide peak using least-squares linear regression and weighting by 1/x. Within day and between days variability measured by a coefficient of variation was <10%. Plasma concentration profiles of normal and tumor bearing rats were compared.
Liquid Scintillation Counting Studies.
Rats were anesthetized with a single i.p. injection of a combination of ketamine (100 mg/kg) and xylazine (2 mg/kg). The neck was shaved and cleaned with surgical disinfectant and alcohol. The right carotid artery was exposed and cannulated with sterile PE-50 tubing. A midline incision was made around the neck region and the left carotid artery was cannulated with PE50 tubing for blood sampling. Catheters were tunneled subcutaneously and exteriorized at the base of the neck followed by closure of incisions using surgical staples. Buehler et al., Free Radic Biol Med 37:124 (2004). The open tubing was stoppered with a fishing line. All surgeries were performed under aseptic conditions. Neosporin antibiotic cream (Pfizer, Morris Plains, N.J.) was applied to the wounds to prevent infection. A 45-minute recovery period was given before drug administration.
IRL1620 was administered i.v. to tumor bearing animals at a dose of 3 nmol/kg. [3H]-paclitaxel (160 μCi/kg) was mixed with unlabeled paclitaxel. Paclitaxel was administered i.v. 15 minutes following vehicle or IRL1620 administration.
Plasma was collected before vehicle or IRL1620 administration to provide baseline values. Approximately, 0.2 mL of blood was drawn from the rats in heparinized syringes at baseline, 1, 5, 15 and 30 minutes and 1, 2, 4, 6, 8, 12 and 24 hours. The samples were centrifuged and plasma was separated and stored at −80° C. until analysis.
The concentrations of [3H]-paclitaxel in the plasma samples were measured using a Beckman Coulter liquid scintillation counter (model LS 6500). Briefly, plasma was thawed and mixed with 20 mL of liquid scintillation cocktail. The samples were counted and the counts were converted from “dpm” units to “fmol/mL” using the following formula:

fmol/mL=dpm value×decay factor×2.2×10−12/10−12×volume of sample in mL

After conversion into fmol/mL, the pharmacokinetics of the total paclitaxel was calculated using the ratio of [3H]-paclitaxel to unlabeled paclitaxel. Plasma paclitaxel pharmacokinetic estimates were determined using both non-compartmental and compartmental analyses as implemented in WinNonlin Pro 4.1 (Pharsight Corp, Mt. View, Calif.).
In the noncompartmental analysis, the area under the curve (AUC0-∞) was estimated using the trapezoidal rule to the last measurable concentration (Clast) and extrapolated to infinity by dividing Clast by the negative value of the terminal slope (λ) of the log-linear plasma concentration vs. time curve. The following parameters were also calculated: mean residence time (MRTiv) was calculated as the reciprocal of λ, systemic clearance (CL) was calculated as the ratio of dose to AUC0-∞ and apparent volume distribution was calculated as the ratio of CL and λ. Plasma half-life was calculated as the product of 0.693 (natural log 2) and MRTiv.
In the compartmental analyses, a series of non-linear compartmental models were fitted to the plasma concentration versus time curve data. Specifically, one-compartmental, two-compartmental and three-compartmental models were compared. Uniform and Predicted data based weighting were tested. The final selection of the model was based on diagnostic plots (observed vs. predicted and plot of residuals), Akaike Information Criteria (AIC) and Schwartz Criteria (SC). The model with a lower AIC and SC criteria was considered the final model.
Data was analyzed by a One Way ANOVA followed by Duncan's test for HPLC-UV studies and by t-test for liquid scintillation studies. A p<0.05 was considered significant. The main outcome measured in these pharmacological response studies was the difference in concentration of paclitaxel in plasma.
The pharmacokinetic profile of paclitaxel was not affected by IRL1620 administration (FIGS. 6 and 7) in normal or tumor bearing rats. HPLC analysis of the plasma pharmacokinetic profile is similar to the more extensive profile of radioactive paclitaxel disposition. FIG. 7 depicts the pharmacokinetic profile of paclitaxel radioactivity in vehicle treated and IRL1620 treated tumor bearing rats. The pharmacokinetic profile was analyzed by noncompartmental and compartmental methods.
In the non-compartmental analysis, the AUC calculated for the vehicle+paclitaxel group was 9433.53±1465.00 ng*h/mL and was similar (p>0.05) to that of IRL1620 treated tumor rats. The elimination half-life was calculated as 0.14±0.08 hour. The clearance calculated as Dose/AUC was estimated to be 0.56±0.07 L/h/kg. The volume of distribution, calculated as clearance/Kel was found to be 10.11±4.17 L/kg. Overall, and as can be seen in the following table, IRL1620 did not affect the pharmacokinetic profile of paclitaxel.















Group
Vehicle + Paclitaxel
IRL1620









Lambda (h)
0.14 ± 0.08
0.10 ± 0.05



Cmax (μg/mL)
6.73 ± 0.54
5.85 ± 0.77



AUC0-∞ (μg-h/mL)
9.43 ± 1.47
8.63 ± 0.79



Cl (L/h/Kg)
0.56 ± 0.07
0.60 ± 0.06



Vd (L/Kg)
10.11 ± 4.18 
9.56 ± 2.90



Vss (L/Kg)
8.14 ± 2.95
8.15 ± 2.20



MRTinf (h)
17.43 ± 8.13 
14.48 ± 4.70 










The plasma concentrations of paclitaxel were calculated from the dpm counts in the plasma samples. A three compartmental model best described the pharmacokinetics of paclitaxel. FIG. 8 depicts the observed versus predicted pharmacokinetic plots for both vehicle treated and IRL1620 treated rats. The AUC of paclitaxel in vehicle treated rats was 9.42±3.18 μg-h/mL. The steady state volume of distribution (Vss) was 10.31±4.54 L/Kg. Clearance was estimated to be 0.69±0.17 L/h/Kg. The αt1⁄2, βt1⁄2γt1⁄2 were 0.03±0.01 hour, 1.0±0.32 hour, and 25.87+17.81 hour, respectively. The mean residence time was 27.92±19.84 hours. As can be seen in the following table, these parameters estimated in the IRL1620 treated group were not significantly different from that in the vehicle treated group.















Group
Vehicle + Paclitaxel
IRL1620









AUC0-∞ (μg-h/mL)
9.42 ± 3.18
7.25 ± 0.75



Cl (L/h/Kg)
0.69 ± 0.17
0.72 ± 0.09



MRT (h)
27.92 ± 19.84
10.58 ± 3.20 



Vss (L/Kg)
10.31 ± 4.54 
7.28 ± 1.79



α t1/2
0.03 ± 0.01
0.04 ± 0.01



β t1/2
 1.0 ± 0.32
0.84 ± 0.32



γ t1/2
25.87 ± 17.81
9.42 ± 2.59



K10
3.14 ± 1.34
1.72 ± 0.57



K12
56.47 ± 27.69
34.93 ± 23.26



K13
5.71 ± 3.37
3.92 ± 1.88










In this study, a three compartmental model best described the plasma pharmacokinetics of paclitaxel. This model suggests that paclitaxel is distributed to various organs whether the blood perfusion in the organs is high, medium or low. IRL1620 administration did not change the distribution of paclitaxel. The plasma pharmacokinetic parameters, generated by the 3-compartment model, displayed comparable clearances, volumes of distribution and absorption, distribution and elimination half-lives for the groups treated with vehicle and IRL1620. However, IRL1620 increases tumor blood perfusion and tumor paclitaxel concentration. Rai et al., American Association of Pharmaceutical Scientists, Pharmaceutics and Drug Delivery Conference. Philadelphia, Pa. (2004); Rai & Gulati, Cancer Chemother Pharmacol, 51:21 (2003). Therefore, IRL1620 selectively increases tumor perfusion without significantly altering the pharmacokinetic profile of paclitaxel.
These studies demonstrated that the use of IRL1620 did not affect the pharmacokinetics of paclitaxel. Often pharmacokinetics can be considered as a surrogate for safety of the compound. Hence these results also suggest that the safety of paclitaxel does not change due to the administration of IRL1620. As a result, IRL1620 could be used to improve paclitaxel efficacy and allow for appropriate dose titration to minimize its severe toxicities.
Example 4
Dose Response Effect of IRL1620, Effect of IRL1620 on the Bio-Distribution of [3H] Paclitaxel in Major Organs and Tumor Tissue and Effect of IRL1620 on Efficacy of Paclitaxel on Tumor Status
The experiments described in the present example were designed to further evaluate (a) the dose response effect of ETB receptor agonist, IRL1620, on breast perfusion of normal and tumor bearing rats, (b) the effect of IRL1620 on the bio-distribution of [3H] paclitaxel in major organs and tumor tissue and (3) the effect of IRL1620 on the efficacy of paclitaxel on tumor status in MNU-induced breast tumor bearing rats.
Virgin female Sprague Dawley rats (Harlan Company, Madison, Wis.) were purchased at 40 days of age and housed two per cage in a temperature-controlled room at 23±1° C. and maintained under a schedule of 12 hourslight/12 hoursdark. They received water and standard rodent diet ad libitum.
IRL1620 was obtained from Sigma Chemical Co. (St. Louis, Mo.). [3H] paclitaxel was purchased from Moravek Biochemicals (Brea, Calif.). Paclitaxel (6 mg/ml solution) was purchased from Ben Venue Laboratories Inc. (Bedford, Ohio). Ketamine and xylazine were purchased from Phoenix Scientific, Inc. (St. Joseph, Mo.). Tissue solubilizer (TS-2) was purchased from RPI Corp. (Chicago, Ill.).
At 48 days of age, each animal received a single i.p. injection of N-methyl nitrosourea (MNU, Ash Stevens, Detroit, Mich.) at a dose of 50 mg/kg. MNU was dissolved in 3% acetic acid and diluted in 0.9% NaCl (final concentration 12.5 mg/ml) and was administered within 30 minutes of preparation. This treatment induces nearly 100% incidence of mammary adenocarcinoma in rats at approximately 100 days of carcinogen treatment. Mehta, Eur J Cancer, 36:1275 (2000). Tumor appearance and location was monitored by manual mammary gland palpation and the tumor surface was measured with a digital caliper. Rats with tumor volume of 500-800 mm3 were selected for the study.
Perfusion Study.
Perfusion to the rat mammary tissue and tumor was measured using a Periflux PF2b 4000 Laser Doppler Flowmetry (Perimed, Stockholm, Sweden) as previously described. Briefly, rats were anesthetized using ketamine (100 mg/kg) and xylazine (2 mg/kg) as a combined single i.p injection. The fur was shaved around the nipples and the animals were placed on a heating pad (37° C.) to minimize temperature variations. The skin surrounding the mammary glands was dissected out at about 6 mm wide and about 4 mm long. A standard model fiber optic probe (MP3 flow probe, Moors Instruments, Devon, England) was applied to the surface of the exposed tissue. It was then connected to a Periflux PF2b 4000 Laser Doppler Flowmetry. The time constant was set to 1.5 seconds and the bandwidth was set to 4 kHz. This method measures a Doppler shift in the laser light (flux), which is determined by erythrocyte number and velocity, and is proportional to the total blood flow with in a given volume of tissue. Flux values were acquired using Polyview software. A 15 minute baseline of stable recording was obtained before the administration of saline or IRL1620. Animals were administered 1, 3 or 9 nmol/kg of IRL1620 and perfusion was recorded for 3 hours. Each dose was administered to at least 4 animals.
The effect of IRL1620 administration on tumor perfusion was found to be transient and dose related (FIG. 9A). A maximum increase of 244.0% (p<0.001) from the baseline in tumor perfusion was observed at 30 minutes after the administration of 3 nmol/kg IRL1620. The increase in perfusion was found to be significant at 15, 30 and 60 minutes compared to baseline as well as saline treated rats. (FIGS. 9A and 9B). Administration of 1 and 9 nmol/kg of IRL1620 produced only marginal increases in breast tumor perfusion compared to the baseline perfusion and that of saline treated rats. Maximum increases in perfusion (60.9 and 63.3%) were recorded at 120 and 30 minutes after 1 and 9 nmol/kg of IRL1620, respectively. However, increases in perfusion in animals treated with 9 nmol/kg of IRL1620 was found to be significant at 15, 30 and 60 minutes as compared to saline treated rats (FIG. 9A). Administration of saline to tumor bearing rats did not produce any significant change in blood perfusion compared to baseline (FIG. 9A). Administration of saline or 1, 3 or 9 nmol/kg IRL1620 did not produce any significant change in breast perfusion in normal female rats (data not shown).
These results show that administration of 3 nmol/kg or 9 nmol/kg IRL1620 produces an increase in tumor perfusion compared to baseline and vehicle treated rats. Although 1, 3 and 9 nmol/kg IRL1620 all increase tumor perfusion somewhat, the 3 nmol/kg dose produces the maximal effect.
Bio-Distribution Study.
Following tumor formation as previously described, rats were anesthetized with ketamine (100 mg/kg) and xylazine (2 mg/kg) as a combined i.p injection. Body weight, tumor location and tumor volume of the rats were documented. The animals were then randomly grouped to receive either saline or IRL1620 (3 nmol/kg) via the tail vain in a final volume of 0.2 ml. Rats from each group then received [3H] paclitaxel (40 μCi/rat in 50:50 of Cremophor EL and ethanol) in a final volume of 1.0 ml at 15, 120 and 240 minutes after IRL1620. Six rats were studied for each time point and a total of 36 rats were used. Animals were sacrificed 3 hours after the administration of [3H] paclitaxel. The concentration of [3H] paclitaxel was determined in the tumor tissue, kidneys, liver, lungs and spleen. Specifically, the tumor and organs were sliced in to small pieces. About 500 mg of the tissue or tumor was placed in separate vials containing tissue solubilizer (6 ml) and incubated in a water bath at 50° C. The vials were removed from the water bath after the tissue or tumor was dissolved and 1.2 ml of 10% glacial acetic acid was added. The contents of the vial were equally divided into 3 vials and 15 ml of liquid scintillation cocktail (Safety Solve, RPI Corp, Chicago, Ill.) was added to each vial and kept overnight for equilibration. The radioactivity in the tubes was counted using a liquid scintillation counter (Beckman Coulter, LS 6500).
The concentration of [3H] paclitaxel in the tumor was significantly increased in IRL1620 (3 nmol/kg) treated rats compared to saline treated rats. The maximal effect was noticed in the group of animals administered paclitaxel 15 minutes after IRL1620 administration. An increase of 277.1, 151:9 and 34.7% in tumor paclitaxel concentration was observed when paclitaxel was administered 15, 120 and 240 minutes, respectively after IRL1620 administration (FIG. 10). IRL1620 administration did not significantly alter the accumulation of paclitaxel in the liver, lungs, kidneys and spleen when compared to control animals (FIG. 10).
Efficacy Study.
Tumor bearing (MNU-treated) animals were randomly divided into seven groups (12 rats/group):

    
    
        Group I—Saline;
        Group II—IRL1620 (3 nmol/kg);
        Group III—Cremophor EL:ethanol;
        Group IV—Vehicle (saline)+paclitaxel (1 mg/kg);
        Group V—Vehicle (saline)+paclitaxel (5 mg/kg);
        Group VI—IRL1620 (3 nmol/kg)+paclitaxel (1 mg/kg); and
        Group VII—IRL1620 (3 nmol/kg)+paclitaxel (5 mg/kg).
    
    


The dosing schedule was once every three days for a total of 5 doses. Body weight, tumor size and location were monitored on every third day for a total of 30 days after the final dose. The following categories were used for scoring: Progression: the tumor grows more than 40% in area compared to commencement of treatment; Stasis: the tumor did not fluctuate more than 40% from its initial area throughout the course of treatment; Partial regression: the tumor regressed more than 40% from its initial area; Complete remission: the tumor is no longer palpable or measurable; Tumor multiplicity: appearance of new tumors during the treatment; and 30 day observation period. The animals were sacrificed 30 days after the final (5th) dose. Data were analyzed using analysis of variance followed by Duncan's test. A level of P<0.05 was considered significant.
Body Weight.
The percentage differences in body weight of animals from baseline (before starting treatment) to 30 days after final dose are given in FIG. 11. The percentage increase in body weight at the end of the experiment in saline treated control rats was 7.2±1.7% compared to baseline body weight. There was a 5.1±3.6, 9.4±2.4, 14.3±3.1 and 13.1±1.8% increase in body weight in the group of animals treated with vehicle+paclitaxel (1 mg/kg), vehicle+paclitaxel (5 mg/kg), IRL1620+paclitaxel 1 mg/kg and IRL1620+paclitaxel 5 mg/kg, respectively (FIG. 11). The percentage increase in body weight of animals administered with Cremophor EL:ethanol and IRL1620 compared to baseline was found to be <10% (data not shown).
Tumor Volume.
Tumor sizes in various groups were comparable and not significantly different from each other at the commencement of treatment (FIG. 12). The tumor volume of control rats increased at a rapid and variable rate. Large variability in tumor growth may be attributed to the random growth pattern of autochthonously growing tumors. At the end of the day observation period, the control tumors had a tumor volume of 2693.4±790.9 mm3. IRL1620 treated rats had a similar pattern of development with a final tumor volume of 2560.5±844.4 mm3. Cremophor EL:Ethanol treatment also resulted in a similar growth pattern with a final tumor volume of 2338±1329 mm3. Thus, IRL1620 and cremophor El:ethanol did not have significant effects on the growth of MNU-induced breast tumors on their own (data not shown). Vehicle+paclitaxel (1 mg/kg) treated rats showed a slightly reduced growth in tumor size (1960.8±611.9 mm3). The vehicle+paclitaxel (5 mg/kg) group showed a larger reduction in tumor volume (1682.7±497.3 mm3) when compared to control rats. IRL1620+paclitaxel (1 mg/kg) treated rats also showed a reduction in tumor size (1707.2±621.1 mm3). However, the lowest average tumor size (730.1±219.4 mm3) was observed in the group of animals treated with IRL1620+paclitaxel (5 mg/kg). IRL1620 followed by 5 mg/kg paclitaxel on every third day for a total of 5 doses significantly (p<0.05) reduced the tumor volume compared to saline+paclitaxel (5 mg/kg) administered rats (FIG. 12). Tumor volume was also found to be lower in IRL1620+paclitaxel (5 mg/kg) group when compared to rats treated with either IRL1620 or cremophor EL:ethanol (data not shown).
Tumor Multiplicity.
Animals in all treatment groups developed additional tumors by the end of 30 day observation period. There was a 58.4, 57.1 and 60.8% increase in additional tumor appearance in animals treated with saline, cremophor EL:ethanol and IRL1620, respectively. New tumor occurrence was found to be 78.3 and 41% in animals administered with vehicle+paclitaxel (1 and 5 mg/kg, respectively). However, percent of additional tumors was found to be 69.2 and 44.8% in IRL1620+paclitaxel (1 and 5 mg/kg, respectively) (data not shown).
Tumor Progression.
The percent of tumors that progressed, remained in stasis, regressed or disappeared were calculated as described previously. 73.5% of tumors in the saline treated group progressed above 40% of the initial tumor size. IRL1620 (82.7%) and cremophor EL:ethanol (80.4%) treated groups had similar percent of tumors progressing past 40% of the initial tumor size. A lower percent of tumors had progressed in the vehicle+paclitaxel (5 mg/kg) group (71.4%), vehicle+paclitaxel (1 mg/kg) group (61.1%) and IRL1620+paclitaxel (1 mg/kg) groups (69%). But, the lowest percent (40%) was seen in the IRL1620+paclitaxel (5 mg/kg) group (FIG. 13).
Tumor Stasis.
16.9% of tumors in the saline treated rats remained in stasis, not growing beyond the 40% range by the 30-day end point. Other control groups showed a slightly lower percentage of tumors remaining in stasis: IRL1620 (13.7%), cremophor EL: ethanol (15.2%). Vehicle+paclitaxel (1 mg/kg) (22.2%) and vehicle+paclitaxel (5 mg/kg) (19.5%) treated rats showed a higher percent of tumors remaining in stasis. IRL1620+paclitaxel (1 mg/kg) (23.8%) treated rats and IRL1620+paclitaxel (5 mg/kg) treated rats showed the greatest percent of tumors remaining in stasis (26.6%) (FIG. 13).
Tumor Regression.
Administration of IRL1620 prior to 5 mg/kg paclitaxel treatment significantly reduced the progression of tumors compared to control animals. The saline treated rats showed 9.2% of tumors regressing from the initial tumor volume. Cremophor EL:ethanol (4.3%), IRL1620 (3.4%) and vehicle+paclitaxel (1 mg/kg) (9.5%) treated rats were not significantly different from the control group in the percent of tumors regressing in size. At the end of 5th dose, tumors had regressed by 76.1±10.5 and 45.9±11.5% in the IRL1620+paclitaxel (5 mg/kg) treated rats and vehicle+paclitaxel (5 mg/kg) treated rats, respectively compared to control rats. There was a 80.2±6.9% (p<0.05) and 33.8±19.4% regression in animals treated with IRL1620+paclitaxel (5 mg/kg) and vehicle+paclitaxel (5 mg/kg) group, respectively (FIG. 13). The tumor regression rate in IRL1620+paclitaxel (1 mg/kg) and vehicle+paclitaxel (1 mg/kg) group was found to be 47.1±15.4 and 37.7±16.2%, respectively. Administration of paclitaxel (5 mg/kg) 15 minutes after IRL1620 produced significantly greater tumor regression compared to administration of paclitaxel (5 mg/kg) 15 minutes after saline. The cremophor El:ethanol and IRL1620 treated groups were not significantly different in their tumor regression compared to the control rats at any point of time (data not shown).
Tumor Remission.
Complete regression, where the tumors completely disappeared, was only observed in two groups. IRL1620+paclitaxel (1 mg/kg) (2.3%) and IRL1620+paclitaxel (5 mg/kg) (15%) treated rats (FIG. 13).
Results of these efficacy studies indicate that administration of IRL1620 significantly increases paclitaxel induced reduction in tumor volume compared to saline treated rats administered with paclitaxel. The enhanced therapeutic benefit seen in the 5 mg/kg dose of paclitaxel was maintained till 30 days after the final dose. This indicates that there was no relapse of the tumor volume and the effect of IRL1620 in enhancing the efficacy of paclitaxel remained consistent till the end of the study. However, saline treatment followed by paclitaxel (1 and 5 mg/kg) did not produce such a significant change in tumor growth. Additionally, tumor multiplicity was reduced in the group of rats treated with IRL1620 followed by paclitaxel (5 mg/kg). Thus, IRL1620 administration prior to paclitaxel (5 mg/kg) has a significant effect on paclitaxel efficacy. This is illustrated by the decrease in tumor burden, percent of tumor regression, unaffected body weight and multiplicity. Further, there was a 2.3 and 15% complete remission of the initial tumors in the group of animals treated with IRL1620 followed by paclitaxel 1 and 5 mg/kg, respectively compared to any other group.
The experiments described in these examples from a breast tumor model clearly show that the ETB receptor agonist, IRL1620 significantly increases tumor blood flow. Administration of IRL1620 produced an increase in tumor blood flow, whereas the perfusion in control healthy tissue was not altered. The increase in tumor perfusion lasted for 3 hours. Administration of [3H] paclitaxel during the window of elevated perfusion significantly increased the concentration of [3H] paclitaxel in the tumor tissue only but not in other organs. Moreover, the results of the experiments described in this example provide evidence that administration of IRL1620 could galvanize the anti-tumor efficacy of paclitaxel. There was a 60.0% reduction in tumor volume of rats treated with paclitaxel (5 mg/kg), every third day for a total of 5 doses, as compared to control rats. However, paclitaxel administration 15 minutes after IRL1620 administration reduced the tumor volume to 268.9% compared to control rats when recorded one month after the last dose of paclitaxel. There was a 130.4% reduction in tumor volume in rats administered IRL1620 as compared to paclitaxel alone treated rats. There is a possibility that the elevated tumor perfusion may increase the availability of nutrients that might facilitate tumor growth. These results show that there was no significant increase in tumor volume and tumor multiplicity of IRL1620 treated rats compared to saline treated rats, indicating that IRL1620 alone did not produce any effect on tumor volume and multiplicity.
PROSTATE TUMOR MODEL
Example 5
Effect of IRL1620 on Prostate Tumor Perfusion, Biodistribution and Efficacy of Doxorubicin and 5-Fluorouracil
After demonstrating that IRL1620 can enhance the efficacy of paclitaxel in a breast tumor model, its effect in a prostate tumor model was also examined. Specifically, whether IRL1620 could enhance the anticancer effects of doxorubicin (DOX) and 5-Fluorouracil (5-FU) in a prostate cancer tumor model was examined.
Five week old Copenhagen prostate tumor bearing rats (Harlan, Indianapolis, Ind.) weighing 100-120 grams were chosen for use in the described prostate tumor model studies. Animal facilities were kept at a controlled temperature (23±1° C.), humidity (50±10%) and on 12 hour light/dark artificial lighting schedule (L0600-1800 h). Rats were housed three to a cage and were given food and water ad libitum. Rats were allowed to acclimate to the environment for at least one week before veterinary examination and the beginning of experimentation. All procedures and animal care were in accordance with the guidelines established by the Animal Care Committee of the University of Illinois at Chicago. Animal facilities were maintained according to Federal Regulations and are accredited by the American Association for Accreditation of Laboratory Animal Care.
IRL1620 was purchased form American Peptide (Sunnyvale, Calif.). [14C]doxorubicin hydrochloride ([14C]adriamycin) was purchased from GE Healthcare (Buckinghamshire, UK). Ketamine and xylazine were purchased from Phoenix Scientific, Inc. (St. Joseph, Mo.). Tissue solubilizer (TS-2) was obtained from RPI Corp. (Chicago, Ill.).
Prostate tumors were induced in male Copenhagen rats using JHU-4 (MAT-LyLu) cells obtained from ATCC (Manassas, Va.). See Gaddipati et al., J Exp Ther Oncol, 4(3): 203-12 (2004) which is hereby incorporated by reference. The cells were maintained in RPMI 1640 medium supplemented with fetal bovine serum (10%) in a humidified incubator containing 5% CO2 at 37° C. Hair was shaved from the dorsal side of the neck, and animals were inoculated with 10,000 JHU-4 (MAT-LyLu) cells in 100 μl phosphate buffered saline by subcutaneous (s.c.) injection. Tumor appearance and location was monitored by manual palpation and tumor diameters were measured with a digital caliper. Experimental procedures began once tumor size reached about 200 mm3.
Perfusion Study.
Rats were anesthetized using ketamine (100 mg/kg) and xylazine (2 mg/kg) as a combined intraperitoneal (i.p.) injection. Fur was shaved around the tumor area and animals were placed on a heating pad (37° C.) to minimize temperature variations. The skin surrounding the tumor tissue was separated out at about 3 mm wide and about 3 mm long to expose the tumor. A standard model fiber optic probe connected to a Periflux PF2b 4000 Laser Doppler Flowmetry (MP3 flow probe, Moors Instruments, Devon, England) was applied to the surface of the exposed tumor. The time constant was set to 1.5 seconds and the bandwidth was set to 4 kHz. This method measures a Doppler shift in the laser light (flux), which is determined by erythrocyte number and velocity, and is proportional to the total blood flow within a given volume of tissue. Flux values were acquired using Polyview software. A 15 minute baseline of stable recording was obtained before the administration of saline or IRL1620. Animals were administered IRL1620 (1, 3, or 6 nmol/kg) in a final volume of 0.2 ml via the tail vein and perfusion was recorded for 3 hours.
As can be seen in FIGS. 14A and 14B, administration of saline or 1 nmol/kg IRL1620 did not produce any significant change in tumor blood perfusion in tumor bearing rats. Administration of 3 nmol/kg or 6 nmol/kg caused a maximal increase in tumor blood perfusion of 102.8% and 79.12% from baseline respectively. This increase in perfusion was significant at 15, 30, and 60 minutes when compared to baseline and saline treated rats (p<0.005). Thus, appropriate doses of IRL1620 transiently increase tumor blood perfusion in an animal model of prostate cancer.
Biodistribution Study.
Tumor bearing rats were randomly grouped (N=6/group) to receive saline or IRL1620 (1, 3 or 6 nmol/kg) via the tail vein in a final volume of 0.2 ml. Rats from each group then received [14C]doxorubicin (1 μCi/rat) i.v. in a final volume of 1.0 ml 15 minutes after saline or IRL1620 administration. Animals were then sacrificed 3 hours after [14C]doxorubicin administration. The concentration of [14C]doxorubicin in the tumor, heart, brain, kidneys, liver, lungs, bone marrow, prostate, skeletal muscles and spleen were examined. Specifically, tumor and organs were sliced into small pieces. The femurs from both legs were separated and weighed with bone marrow flushed out using a syringe containing tissue solubilizer. About 500 mg of tissue or tumor was placed in separate vials containing tissue solubilizer (6 ml) and incubated in a water bath at about 50° C. The vials were removed from the water bath after the tissue or tumor was dissolved and 1.2 ml of 10% glacial acetic acid was added. The contents of the vial were then equally divided into 3 vials and 15 ml of liquid scintillation cocktail (Safety Solve, RPI Corp, Chicago, Ill.) was added to each vial and kept overnight for equilibration. The radioactivity in the tubes was counted using a liquid scintillation counter (Beckman Coulter, LS 6500).
As can be seen in FIG. 15, administration of 1 nmol/kg IRL1620 did not produce any significant change in the uptake of [14C]doxorubicin in tumor or other tissues. Concentrations of [14C]doxorubicin in tumor, however, were significantly increased in those rats receiving 3 nmol/kg IRL1620 (115.85% increase; p<0.01) or 6 nmol/kg IRL1620 (80.02% increase; p<0.05) when compared to saline treated rats. No dose of IRL1620 produced significant increases in the accumulation of [14C]doxorubicin in the heart, brain, kidneys, liver, lungs, bone marrow, prostate, skeletal muscles or spleen when compared to control animals. Thus, IRL1620 can selectively increase delivery of chemotherapeutic agents to tumor tissue.
Efficacy Studies: Effect of IRL1620 on the Efficacy of Doxorubicin (DOX)
In this study, IRL1620 (N-Succinyl-[Glu9,Ala11,15] Endothelin fragment 8-21) was obtained from American Peptides (Sunnyvale, Calif.). Doxorubicin hydrochloride (adriamycin, 2 mg/ml solution) was purchased from Ben Venue Laboratories Inc. (Bedford, Ohio). 5-Fluorouracil (50 mg/ml) was purchased from Cadlia Pharmaceuticals (Ahmedabad, India). Ketamine and xylazine were purchased from Phoenix Scientific, Inc. (St. Joseph, Mo.).
Prostate tumors were induced in five week old male Copenhagen rats (Harlan, Indianapolis, Ind.) as previously described. Experimental procedures began once tumor size reached about 200 mm3. Tumor bearing animals were randomly divided into six groups (8 rats/group):

    
    
        Group I—Saline;
        Group II—IRL1620 (3 nmol/kg);
        Group III—Vehicle (saline)+DOX (2.5 mg/kg);
        Group IV—Vehicle (saline)+DOX (5 mg/kg);
        Group V—IRL1620 (3 nmol/kg)+DOX (2.5 mg/kg); and
        Group VI—IRL1620 (3 nmol/kg)+DOX (5 mg/kg).
    
    


DOX was diluted in saline in a final volume of 1.0 ml and injected via the tail vein 15 minutes after saline or IRL1620 administration. The dosing schedule was once every three days for a total of 4 doses. Body weight and tumor size were monitored on every third day up to 12 days after the final dose. The animals were sacrificed 12 days after the final (4th) dose. Tumors were separated and weighed, and tissues were observed for gross metastasis. Tissues and tumor were preserved in 10% buffered formalin for histopathological analysis.
Body Weight.
There was an increase in body weight in the groups of rats treated with saline or IRL1620 alone. The body weight of saline or IRL1620 alone animals in the beginning of the experiment were found to be 152±6.97 and 148.8±2.52 g respectively which increased to 178.8±4.7 and 175.72±2.35 g, respectively, on day 19 of the study. Rats receiving saline and DOX, or IRL1620 and DOX showed a decrease in body weight. The maximum decrease in body weight was found to be −8.46±3.88 and −10.03±2.12 in the group of rats treated with saline+DOX (5 mg/kg) and IRL1620+DOX (5 mg/kg), respectively, on day 13. However, the decrease in body weight was not found to be significant at any time point between any of the groups administered with DOX from the beginning of treatment (FIG. 16A).
Tumor Volume.
Tumor volume of saline or IRL1620 treated rats increased at a rapid rate and all rats in these groups were sacrificed on day 19 due to large tumor burden. All other groups were sacrificed on day 22. The saline or IRL1620 treated rats had a tumor volume of 10166±957 and 11033±873 mm3, respectively, upon sacrifice. The tumor volume of saline+DOX (2.5 mg/kg) and saline+DOX (5 mg/kg) treated rats was found to be 9102±1442 and 4204±299 mm3, respectively. IRL1620+DOX (2.5 mg/kg) treated rats recorded a tumor volume of 5544±845 mm3. The lowest tumor volume (1965±332 mm3) was observed in the group of rats treated with IRL1620+DOX (5 mg/kg). The decrease in tumor volume in rats treated with IRL1620+DOX (5 mg/kg) was found to be significant on days 10, 13, 16, 19 and 22 as compared to saline+DOX (5 mg/kg) treated rats (FIG. 16B).
Tumor Weight.
Saline, IRL1620, and saline+DOX (2.5 mg/kg) treated rats had a comparable tumor weight upon sacrifice (19.14±1.8, 20.77±1.64 and 17.14±2.42 g, respectively). The tumor weight or rats treated with saline+DOX (5 mg/kg); IRL1620+DOX (2.5 mg/kg) and IRL1620+DOX (5 mg/kg) was reduced to 7.19±0.56, 10.44±1.42 and 3.31±1.64 g respectively upon sacrifice on day 22. There was a significant difference in the tumor weight between saline+DOX (5 mg/kg) and IRL1620+DOX (5 mg/kg) treated rats (p<0.005). These studies demonstrate that IRL1620 significantly increased the anticancer efficacy of DOX (FIG. 16C).
Efficacy Studies: Effect of IRL1620 on the Efficacy of 5-Fluorouracil (5-FU)
In a follow up study, the same procedures were followed except that tumor bearing rats were randomized into the following six groups (6 rats/group) to examine the effect of IRL1620 on the efficacy of 5-FU:

    
    
        Group I—Saline;
        Group II—IRL1620 (3 nmol/kg);
        Group III—Vehicle (saline)+5-FU (25 mg/kg);
        Group IV—Vehicle (saline)+5-FU (50 mg/kg);
        Group V—IRL1620 (3 nmol/kg)+5-FU (25 mg/kg); and
        Group VI—IRL1620 (3 nmol/kg)+5-FU (50 mg/kg).
    
    


5-FU was diluted in saline in a final volume of 1.0 ml and injected via tail vein 15 minutes after saline or IRL1620 administration. The dosing schedule and procedures were same as that of the above-mentioned DOX study.
Body Weight.
The body weight of saline or IRL1620 animals on the beginning of the experiment was found to be 172±4.87 and 176.9±6.19 g respectively, which was increased to 200.3±2.57 and 202.2±7.28 g, respectively on day 16. The percent difference in body weight gain of saline or IRL1620 animals on day 16 from the beginning of the experiment was found to be 16.17±1.64 and 14.19±1.66 respectively. The body weight of rats in other groups were comparable on the onset of treatment and were found to be 170.38±2.61, 174.6±3.45, 174.7±5.78 and 179.45±2.53 g for saline+5-FU (25 mg/kg), saline+5-FU (50 mg/kg), IRL1620+5-FU (25 mg/kg) and IRL1620+5-FU (50 mg/kg) respectively. However, body weight declined during by the following amounts: saline+5-FU (25 mg/kg): −5.46±3.39; saline+5-FU (50 mg/kg): −10.97±2.18; IRL1620+5-FU (25 mg/kg): −8.27±2.31; and IRL1620+50 mg/kg: −11.20±2.41 (FIG. 17A).
Tumor Volume.
Saline or IRL1620 treated rats had a rapid progressive increase in tumor size and all rats in these groups were sacrificed on day 16 due to large tumor burden. There was no significant difference in the tumor size of rats administered with saline+5-FU (25 mg/kg) and IRL1620+5-FU (25 mg/kg). However, there was a consistent significant decrease in tumor volume of rats treated with IRL1620+5-FU (50 mg/kg) on days 13, 16, 19 and 22 as compared to saline+FU (50 mg/kg) treated rats (FIG. 17B).
Tumor Weight.
There were no significant differences in tumor weights of rats administered with saline or IRL1620 upon sacrifice on day 16 (weights were 17.92±2.01 and 19.50±2.37 g respectively). There was a 38.18% decrease in the tumor weight of rats treated with IRL1620+5-FU (25 mg/kg) as compared to saline+5-FU (25 mg/kg) treated animals. Moreover, there was a 167.19% difference in tumor weight between IRL1620+5-FU (50 mg/kg) and saline+5-FU (50 mg/kg) treated rats (p<0.01) (FIG. 17C). These results demonstrate that IRL1620 significantly increases the efficacy of 5-FU in reducing tumor volume and tumor weight.
These studies collectively demonstrate that IRL1620 is effective in an animal model of prostate cancer to enhance tumor blood perfusion, increase delivery of chemotherapeutic agents to the tumor and enhance the efficacy of chemotherapeutic agents.
MELANOMA MODEL
Example 6
Effect of IRL1620 on Tumor Perfusion and Biodistribution of Paclitaxel
Male nude mice were used in the described melanoma model studies. All procedures and animal care were in accordance with the guidelines established by the Animal Care Committee of the University of Illinois at Chicago. Animal facilities were kept at a controlled temperature (23±1° C.) humidity (50±10%) and on artificial lighting (L0600-1800 h). Animal facilities were maintained according to Federal Regulations and are accredited by the American Association for Accreditation of Laboratory Animal Care.
IRL1620 (N-Succinyl-[Glu9,Ala11,15] Endothelin fragment 8-21) was obtained from Sigma Chemical Co. (St. Louis, Mo.). [3H]paclitaxel was purchased from Moravek Biochemicals (Brea, Calif.). Paclitaxel (6 mg/ml solution) was purchased from Ben Venue Laboratories Inc. (Bedford, Ohio). Ketamine and xylazine were purchased from Phoenix Scientific, Inc. (St. Joseph, Mo.). Tissue solubilizer (TS-2) was obtained from RPI Corp. (Chicago, Ill.).
A cell line inoculated transplanted melanoma model was used for the study. Mice were subcutaneously inoculated with one million human melanoma cells (UISO-MEL-2). Mice with a tumor volume of about 200-400 mm3 were selected for the study.
Perfusion Study.
Mice (N=4/group) were anesthetized using ketamine (150 mg/kg) and xylazine (2 mg/kg) as a combined i.p injection. Animals were placed on a heating pad (37° C.) to minimize temperature variations. A 10 mm long incision was made on the skin surrounding the tumor. A standard model fiber optic probe connected to a Periflux PF2b 4000 Laser Doppler Flowmetry (MP3 flow probe, Moors Instruments, Devon, England) was applied to the surface of the exposed tumor. The time constant was set to 1.5 seconds and the bandwidth was set to 4 kHz. Flux values were acquired using Polyview software. A 15 minute baseline of stable recording was obtained before the administration of saline or IRL1620 (3 nmol/kg) via tail vein and perfusion was recorded for 3 hours.
Administration of saline did not produce any significant change in tumor blood perfusion of melanoma mice. An increase of 154.4%, 189.0%, 198.1%, 172.8% and 94.07.12% from baseline in tumor perfusion was observed at 30, 60, 90, 120 and 150 minutes respectively following IRL1620 administration. Thus, IRL1620 significantly increased tumor blood perfusion in melanoma mice when compared to saline-treated controls. This effect was transient, lasting for about 2 hours (FIGS. 18A and 18B).
Biodistribution Study.
Tumor bearing mice were anesthetized with ketamine (150 mg/kg) and xylazine (2 mg/kg) as a combined i.p injection. The animals were randomly grouped (N=4/group) to receive saline or IRL1620 (3 nmol/kg) via the tail vein in a final volume of 0.1 ml. Mice from each group also received [3H]paclitaxel (10 μCi/mice in 50:50 of Cremophor EL and ethanol) diluted to a concentration of 20:80 [3H]paclitaxel:saline in a final volume of 1.0 ml (i.v) 15 minutes after saline or IRL1620 administration. Animals were sacrificed 3 hours after the administration of [3H]paclitaxel. The concentration of [3H]paclitaxel was determined in the tumor, heart, kidneys, liver, lungs and spleen. The tumor and organs were sliced in to small pieces. 500 mg of tissue or tumor was placed in separate vials containing tissue solubilizer (6 ml) and incubated in a water bath at 50° C. The vials were removed from the water bath after the tissue or tumor was dissolved and 1.2 ml of 10% glacial acetic acid was added. The content of the vial were equally divided into 3 vials and 15 ml of liquid scintillation cocktail (Safety Solve, RPI Corp, Chicago, Ill.) was added to each vial and kept overnight for equilibration. The radioactivity in the tubes was counted using a liquid scintillation counter (Beckman Coulter, LS 6500).
As shown in FIG. 19, tumor [3H]paclitaxel concentration was significantly increased in IRL1620 treated mice compared to saline treated mice. Tumor [3H]paclitaxel concentration was found to be 40.77 and 274.28 nmol/g tissue in animals injected with saline or IRL1620, respectively, 15 minutes before [3H]paclitaxel administration. There was a 572.99% increase in tumor [3H]paclitaxel of animals treated with IRL1620 compared to vehicle treated mice. However, IRL1620 administration did not produce significant increases in the accumulation of [3H]paclitaxel in the heart, kidneys, liver, lungs or spleen when compared to saline treated animals. Thus, IRL1620 can significantly enhance the uptake and delivery of paclitaxel to tumor tissues without affecting its delivery to other organs.
In conclusion, IRL1620 can be used as a tumor-selective vasodilator and can be used to selectively increase the delivery and efficacy of chemotherapeutic agents. The present study clearly demonstrates that multi-fold higher drug concentrations can be achieved in the tumor tissue by adopting this therapeutic strategy. Finally, ETA receptor antagonists have also been proposed to improve tumor blood flow (Sonveaux et al., Cancer Res, 64:3209 (2004)) and can be used to enhance delivery of anticancer drugs to the tumor in accordance with the present invention.
Pharmaceutical compositions containing the described active ingredients are suitable for administration to humans or other mammals. Typically, the pharmaceutical compositions are sterile, and contain no toxic, carcinogenic, or mutagenic compounds that would cause an adverse reaction when administered. Administration of the pharmaceutical composition can be performed before, during, or after the onset of solid tumor growth.
A method of the present invention can be accomplished using active ingredients as described above, or as a physiologically acceptable salt, derivative, prodrug, or solvate thereof. The active ingredients can be administered as the neat compound, or as a pharmaceutical composition containing either or both entities.
The pharmaceutical compositions include those wherein the active ingredients are administered in an effective amount to achieve their intended purpose. More specifically, a “therapeutically effective amount” means an amount effective to prevent development of, to eliminate, to retard the progression of, or to reduce the size of a solid tumor. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
A “therapeutically effective dose” refers to that amount of the active ingredients that results in achieving the desired effect. Toxicity and therapeutic efficacy of such active ingredients can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. A high therapeutic index is preferred. The data obtained can be used in formulating a range of dosage for use in humans. The dosage of the active ingredients preferably lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized.
The exact formulation and dosage is determined by an individual physician in view of the patient's condition. Dosage amount and interval can be adjusted individually to provide levels of the active ingredients that are sufficient to maintain therapeutic or prophylactic effects.
The amount of pharmaceutical composition administered can be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.
The active ingredients can be administered alone, or in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present invention thus can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active ingredients into preparations which can be used pharmaceutically.
When a therapeutically effective amount of the active ingredients is administered, the composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred composition for intravenous injection typically will contain an isotonic vehicle although this characteristic is not required.
For veterinary use, the active ingredients are administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian can readily determine the dosing regimen that is most appropriate for a particular animal.
Various adaptations and modifications of the embodiments can be made and used without departing from the scope and spirit of the present invention which can be practiced other than as specifically described herein. The above description is intended to be illustrative, and not restrictive. The scope of the present invention is to be determined only by the claims.
The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described, or portions thereof, it being recognized that various modifications are possible within the scope of the present invention claimed. Moreover, any one or more features of any embodiment of the present invention can be combined with any one or more other features of any other embodiment of the present invention, without departing from the scope of the present invention.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
The terms “a” and “an” and “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the present invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present invention.
Groupings of alternative elements or embodiments of the present invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these certain embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety.
In closing, it is to be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the present invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

## Claims

1. An article of manufacture comprising: (a) a packaged composition comprising an IRL-1620, (b) a packaged composition comprising a chemotherapeutic agent; (c) an insert providing instructions for the administration of (a) and (b) for the treatment of a solid tumor in a mammal, wherein said solid tumor is a prostate tumor or a melanoma tumor; and (d) a container for (a), (b), and (c).

2. The article of manufacture according to claim 1, wherein said chemotherapeutic agent is selected from the group consisting of adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, and mixtures thereof.

3. The article of manufacture according to claim 1, wherein said chemotherapeutic agent is paclitaxel.

4. An article of manufacture comprising: (a) a packaged composition comprising an IRL-1620; (b) a packaged composition comprising a chemotherapeutic agent; (c) an insert providing instructions for a simultaneous or sequential administration of (a) and (b) to treat a solid tumor in a mammal, wherein said solid tumor is a prostate tumor or a melanoma tumor; and (d) a container for (a), (b), and (c).

5. The article of manufacture according to claim 4, wherein said chemotherapeutic agent is selected from the group consisting of adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, and mixtures thereof.

6. The article of manufacture according to claim 4, wherein said chemotherapeutic agent is selected from the group consisting of paclitaxel, doxorubicin, 5-fluorouracil, and combinations thereof.

7. The article of manufacture according to claim 4, wherein said chemotherapeutic agent is paclitaxel.

8. The article of manufacture according to claim 4, wherein said chemotherapeutic agent is cisplatin.

9. An article of manufacture comprising a composition comprising an IRL-1620 and a chemotherapeutic agent, and instructional information directing the administration of said composition with a chemotherapeutic agent to treat a solid tumor, wherein said solid tumor is a prostate tumor or a melanoma tumor.

10. The article of manufacture according to claim 9, wherein said chemotherapeutic agent is selected from the group consisting of adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, 5-fluorouracil, and combinations thereof.

11. The article of manufacture according to claim 9, wherein said chemotherapeutic agent is selected from the group consisting of paclitaxel, doxorubicin, 5-fluorouracil, and combinations thereof.

12. The article of manufacture according to claim 9, wherein said chemotherapeutic agent is paclitaxel.

13. The article of manufacture according to claim 9, wherein said chemotherapeutic agent is cisplatin.

14. An article of manufacture comprising: (a) a packaged composition comprising an IRL-1620; (b) a packaged composition comprising a chemotherapeutic agent; (c) an insert providing instructions for administration of (a) and (b) to treat a prostate tumor in a mammal; and (d) a container for (a), (b), and (c).

15. An article of manufacture comprising: (a) a packaged composition comprising an IRL-1620; (b) a packaged composition comprising a paclitaxel; (c) an insert providing instructions for a simultaneous or sequential administration of (a) and (b) to treat a prostate tumor in a mammal; and (d) a container for (a), (b), and (c).

16. An article of manufacture comprising: (a) a packaged composition comprising an IRL-1620; (b) a packaged composition comprising a chemotherapeutic agent; (c) an insert providing instructions for administration of (a) and (b) to treat a melanoma tumor in a mammal, and (d) a container for (a), (b), and (c).

17. An article of manufacture comprising: (a) a packaged composition comprising an IRL-1620; (b) a packaged composition comprising a cisplatin; (c) an insert providing instructions for a simultaneous or sequential administration of (a) and (b) to treat a melanoma tumor in a mammal; and (d) a container for (a), (b), and (c).